The Principal Investigator seeks advanced training in a mentored environment to study the regulation of cardiac morphogenesis by Nkx genes in the zebrafish embryo. In the laboratory of Dr. Deborah Yelon and with the support of the Division of Pediatric Cardiology at Columbia University, the primary objective is to provide the Principal Investigator with an environment to pursue training in formal courses and laboratory research that will enhance her development into an independent physician-scientist. The Principal Investigator will use the zebrafish model to study defects in cardiac chamber formation as a means to understand the genetic regulation of congenital heart disease (CHD). NKX2-5 is a key causative gene in human CHD and plays a critical role in normal chamber formation in mice. However, comprehension of the underlying cellular and molecular mechanisms regulated by NKX2-5 is extremely limited. Our preliminary studies suggest that loss-of-function of the zebrafish NKX2-5 homologs, nkx2.5 and nkx2.7, impairs morphogenesis of the ventricular and atrial cardiac chambers. Furthermore, we find that the first sign of abnormal cardiac morphogenesis in these embryos appears during the initial assembly of the heart: specifically, the inhibition of nkx2.5 and nkx2.7 gene function leads to the formation of an abnormally short and wide ventricular portion of the heart tube. We hypothesize that nkx genes are critical regulators of the cellular processes that drive the early stages of heart tube assembly. To test this hypothesis, we propose three specific aims: 1. To characterize the cellular mechanisms by which nkx genes regulate elongation of the developing ventricle. Through phenotypic characterization of nkx-deficient embryos, we will test the hypothesis that nkx genes guide specific aspects of proliferation, cell shape change, cell movement, and/or cell polarity during the process of ventricle assembly. 2. To define the cellular role that nkx genes play in atrial morphogenesis. Having determined the impact of nkx genes on ventricular morphogenesis, we will use similar experimental strategies to test the hypothesis that nkx genes play a parallel role during atrial assembly. 3. To identify the downstream effector genes through which nkx genes regulate heart tube assembly. Using microarray analysis and subsequent validation, annotation, and loss-of-function analysis of target genes, we will identify the components of the nkx pathway that play essential roles during the initial assembly of the heart. Together, these studies will shed new light on the cellular processes and molecular mechanisms underlying CHD. In the long term, this insight may provide us with the opportunity to generate better prognostic information, improved prediction of recurrence risk, and possible disease prevention within families with CHD. (End of Abstract)